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Local Inhibition of MEK/Akt Prevents Cellular Growth in Human Congenital Melanocytic Nevi

  • Author Footnotes
    10 These authors contributed equally to this work.
    Thomas Rouillé
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Saint-Antoine Research Center, INSERM UMRS_938, Paris, France

    Sorbonne Université, Paris, France
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  • Author Footnotes
    10 These authors contributed equally to this work.
    Selim Aractingi
    Footnotes
    10 These authors contributed equally to this work.
    Affiliations
    Saint-Antoine Research Center, INSERM UMRS_938, Paris, France

    Université Paris-Descartes, Paris, France

    AP-HP, Hôpital Cochin, Department of Dermatology, Paris, France
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  • Natacha Kadlub
    Affiliations
    Université Paris-Descartes, Paris, France

    AP-HP, Hôpital Necker-Enfants-Malades, Department of Maxillofacial and Plastic Surgery, Paris, France
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  • Sylvie Fraitag
    Affiliations
    AP-HP, Hôpital Necker-Enfants-Malades, Department of Pathology, Paris, France
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  • Alexandre How-Kit
    Affiliations
    Laboratory for Functional Genomics, Fondation Jean Dausset–CEPH, Paris, France
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  • Antoine Daunay
    Affiliations
    Laboratory for Functional Genomics, Fondation Jean Dausset–CEPH, Paris, France
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  • Mikael Hivelin
    Affiliations
    Université Paris-Descartes, Paris, France

    AP-HP, Hôpital Européen Georges-Pompidou, Department of Plastic Surgery, Paris, France
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  • Philippe Moguelet
    Affiliations
    AP-HP, Hôpital Tenon, Department of Pathology, Paris, France
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  • Arnaud Picard
    Affiliations
    Université Paris-Descartes, Paris, France

    AP-HP, Hôpital Necker-Enfants-Malades, Department of Maxillofacial and Plastic Surgery, Paris, France
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  • Author Footnotes
    11 These authors share senior authorship. This work has been performed in Paris, Ile de France, France.
    Romain H. Fontaine
    Footnotes
    11 These authors share senior authorship. This work has been performed in Paris, Ile de France, France.
    Affiliations
    Saint-Antoine Research Center, INSERM UMRS_938, Paris, France

    Sorbonne Université, Paris, France
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  • Author Footnotes
    11 These authors share senior authorship. This work has been performed in Paris, Ile de France, France.
    Sarah Guégan
    Correspondence
    Correspondence: Sarah Guégan, INSERM UMRS_938, Saint-Antoine Research Center, Sorbonne Université, 27 Rue de Chaligny, 75012 Paris, France.
    Footnotes
    11 These authors share senior authorship. This work has been performed in Paris, Ile de France, France.
    Affiliations
    Saint-Antoine Research Center, INSERM UMRS_938, Paris, France

    Université Paris-Descartes, Paris, France

    AP-HP, Hôpital Cochin, Department of Dermatology, Paris, France
    Search for articles by this author
  • Author Footnotes
    10 These authors contributed equally to this work.
    11 These authors share senior authorship. This work has been performed in Paris, Ile de France, France.
Open ArchivePublished:May 03, 2019DOI:https://doi.org/10.1016/j.jid.2019.03.1156
      The management of large congenital melanocytic nevi (lCMN) is based exclusively on iterative surgical procedures in the absence of validated medical therapy. The aim of our study was to develop an intra-lesional medical treatment for lCMN. Seventeen patients harboring NRAS-mutated lCMN were included. Nevocytes obtained from lCMN displayed an overactivation of mitogen-activated protein kinase and phosphoinositide 3-kinase (Akt) pathways. Mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK) and Akt inhibitors reduced the nevosphere diameter in sphere-forming assays, as well as cell viability and proliferation in in vitro assays. Standardized lCMN explants were then cultured ex vivo with the same inhibitors, which induced a decrease in MelanA+ and Sox10+ cells in both epidermis and dermis. Finally, intradermal injections of these inhibitors were administered within standardized lCMN xenografts in Rag2–/– mice. They induced a dramatic decrease in nevocytes in treated xenografts, which persisted 30 days after the end of treatment. Using original nevus explant and xenograft preclinical models, we demonstrated that intradermal MEK/Akt inhibition might serve as neoadjuvant therapy for the treatment of NRAS-mutated congenital melanocytic nevi to avoid iterative surgeries.

      Abbreviations:

      Akt (phosphoinositide 3-kinase), CMN (congenital melanocytic nevi), ERK (extracellular signal–regulated kinase), GSK (glycogen synthase kinase), K14 (keratin 14), lCMN (large congenital melanocytic nevi), MAPK (mitogen-activated protein kinase), MEK (MAPK/ERK kinase), PI3K (phosphatidylinositol 3-kinase), TBST (Tween 20)

      Introduction

      Human congenital melanocytic nevi (CMN) can be associated with life-threatening complications (
      • Alikhan A.
      • Ibrahimi O.A.
      • Eisen D.B.
      Congenital melanocytic nevi: where are we now? Part I. Clinical presentation, epidemiology, pathogenesis, histology, malignant transformation, and neurocutaneous melanosis.
      ,
      • Castilla E.E.
      • da Graça Dutra M.
      • Orioli-Parreiras I.M.
      Epidemiology of congenital pigmented naevi: I. Incidence rates and relative frequencies.
      ,
      • Vourc’h-Jourdain M.
      • Martin L.
      • Barbarot S.
      aRED
      Large congenital melanocytic nevi: therapeutic management and melanoma risk: a systematic review.
      ). Large CMN (lCMN) and giant CMN, defined by a projected adult size ranging from 20 cm to 40 cm or > 40 cm, respectively, are at an increased risk of malignant transformation in cutaneous and central nervous system melanomas (
      • Gerami P.
      • Paller A.S.
      Making a mountain out of a molehill: NRAS, mosaicism, and large congenital nevi.
      ,
      • Krengel S.
      • Hauschild A.
      • Schäfer T.
      Melanoma risk in congenital melanocytic naevi: a systematic review.
      ,
      • Krengel S.
      • Reyes-Múgica M.
      Melanoma risk in congenital melanocytic naevi.
      ,
      • Lu C.
      • Zhang J.
      • Nagahawatte P.
      • Easton J.
      • Lee S.
      • Liu Z.
      • et al.
      The genomic landscape of childhood and adolescent melanoma.
      ,
      • Marghoob A.A.
      • Schoenbach S.P.
      • Kopf A.W.
      • Orlow S.J.
      • Nossa R.
      • Bart R.S.
      Large congenital melanocytic nevi and the risk for the development of malignant melanoma. A prospective study.
      ,
      • Swerdlow A.J.
      • English J.S.
      • Qiao Z.
      The risk of melanoma in patients with congenital nevi: a cohort study.
      ). They can be associated with extracutaneous manifestations such as diffuse leptomeningeal melanocytic infiltration (i.e., neurocutaneous melanocytosis) (
      • Kadonaga J.N.
      • Frieden I.J.
      Neurocutaneous melanosis: definition and review of the literature.
      ,
      • Ruiz-Maldonado R.
      • del Rosario Barona-Mazuera M.
      • Hidalgo-Galván L.R.
      • Medina-Crespo V.
      • Duràn-Mckinster C.
      • Tamayo-Sánchez L.
      • et al.
      Giant congenital melanocytic nevi, neurocutaneous melanosis and neurological alterations.
      ). The mainstay of CMN treatment is surgical removal, lCMN usually requiring iterative surgical procedures along with possible complications (
      • Ibrahimi O.A.
      • Alikhan A.
      • Eisen D.B.
      Congenital melanocytic nevi: where are we now? Part II. Treatment options and approach to treatment.
      ). Moreover, multiple resections of lCMN and giant CMN do not prevent melanoma emergence even if cosmesis is improved (
      • Mérigou D.
      • Prey S.
      • Niamba P.
      • Loot M.
      • Lepreux S.
      • Boralevi F.
      • et al.
      Management of congenital nevi at a dermatologic surgical paediatric outpatient clinic: consequences of an audit survey 1990–1997.
      ). Therefore, there is a need for new medical options.
      Previous studies have shown that 80% of CMN harbor mutations affecting NRAS exon 3 (
      • Bauer J.
      • Curtin J.A.
      • Pinkel D.
      • Bastian B.C.
      Congenital melanocytic nevi frequently harbor NRAS mutations but no BRAF mutations.
      ,
      • Charbel C.
      • Fontaine R.H.
      • Malouf G.G.
      • Picard A.
      • Kadlub N.
      • El-Murr N.
      • et al.
      NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.
      ,
      • Ichii-Nakato N.
      • Takata M.
      • Takayanagi S.
      • Takashima S.
      • Lin J.
      • Murata H.
      • et al.
      High frequency of BRAFV600E mutation in acquired nevi and small congenital nevi, but low frequency of mutation in medium-sized congenital nevi.
      ,
      • Salgado C.M.
      • Basu D.
      • Nikiforova M.
      • Bauer B.S.
      • Johnson D.
      • Rundell V.
      • et al.
      BRAF mutations are also associated with neurocutaneous melanocytosis and large/giant congenital melanocytic nevi.
      ). The most frequent amino acid substitutions (Q61K and Q61R) result in the synthesis of an aberrant protein unable to hydrolyze guanosine triphosphate, causing it to be constitutively active (
      • Takata M.
      • Saida T.
      Genetic alterations in melanocytic tumors.
      ). Downstream signaling pathways, such as Raf-mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) kinase (MEK)-ERK, phosphatidylinositol 3-kinase (PI3K)- protein kinase B (Akt)/pyruvate dehydrogenase kinase 1, Ral guanine nucleotide dissociation stimulator, and phospholipase C/protein kinase C pathways are thus activated, with their dysregulation leading to increased cell survival and proliferation (
      • Samatar A.A.
      • Poulikakos P.I.
      Targeting RAS-ERK signalling in cancer: promises and challenges.
      ). Efficient inhibitors directly targeting NRAS are not yet available. However,
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      reported the anti-tumor activity of combined targeting of MEK/ERK and PI3K/mTOR pathways on NRAS-mutated melanoma both in vitro and in vivo. In addition, MEK inhibitors allow partial and transient control of tumor growth in patients displaying NRAS-mutated metastatic melanoma (
      • Ascierto P.A.
      • Schadendorf D.
      • Berking C.
      • Agarwala S.S.
      • van Herpen C.M.
      • Queirolo P.
      • et al.
      MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study.
      ). The use of MEK inhibitors, binimetinib or trametinib, on a compassionate basis has also been reported in children harboring NRAS-mutated central nervous system melanoma or neurocutaneous melanocytosis associated with CMN (
      • Kinsler V.A.
      • O’Hare P.
      • Jacques T.
      • Hargrave D.
      • Slater O.
      MEK inhibition appears to improve symptom control in primary NRAS-driven CNS melanoma in children.
      ,
      • Küsters-Vandevelde H.V.
      • Willemsen A.E.
      • Groenen P.J.
      • Küsters B.
      • Lammens M.
      • Wesseling P.
      • et al.
      Experimental treatment of NRAS-mutated neurocutaneous melanocytosis with MEK162, a MEK-inhibitor.
      ). Furthermore, Basu and colleagues (2016) cultured cells from 4 patients with malignant neurocutaneous melanocytosis and showed that drugs targeting the MAPK signaling pathway downstream of NRAS reduced cell viability.
      In CMN, however, medical treatments have not yet been tested. The aim of our study was, therefore, to develop alternative models of study to assess the effect of local treatments targeting the NRAS-driven signaling pathways in CMN. Indeed, similar local medical therapies have already been evaluated in other cutaneous tumors. Topical mTOR inhibitors, rapamycin and sirolimus, have been used for the treatment of angiofibromas in tuberous sclerosis complex (
      • Balestri R.
      • Neri I.
      • Patrizi A.
      • Angileri L.
      • Ricci L.
      • Magnano M.
      Analysis of current data on the use of topical rapamycin in the treatment of facial angiofibromas in tuberous sclerosis complex.
      ,
      • Malissen N.
      • Vergely L.
      • Simon M.
      • Roubertie A.
      • Malinge M.C.
      • Bessis D.
      Long-term treatment of cutaneous manifestations of tuberous sclerosis complex with topical 1% sirolimus cream: a prospective study of 25 patients.
      ). Topical imiquimod has been tested in the neoadjuvant setting in basal cell carcinoma (
      • Jansen M.H.E.
      • Mosterd K.
      • Arits A.H.M.M.
      • Roozeboom M.H.
      • Sommer A.
      • Essers B.A.B.
      • et al.
      Five-year results of a randomized controlled trial comparing effectiveness of photodynamic therapy, topical imiquimod, and topical 5-fluorouracil in patients with superficial basal cell carcinoma.
      ;
      • Shaw F.M.
      • Weinstock M.A.
      Comparing topical treatments for basal cell carcinoma.
      ). Oral MEK inhibitors have been investigated in nonmalignant cutaneous tumors, such as inoperable plexiform neurofibromas in type 1 neurofibromatosis (
      • Dombi E.
      • Baldwin A.
      • Marcus L.J.
      • Fisher M.J.
      • Weiss B.
      • Kim A.
      • et al.
      Activity of selumetinib in neurofibromatosis Type 1-related plexiform neurofibromas.
      ). Therefore, we set up 2 preclinical models displaying the main characteristics of the human condition and tested various targeted inhibitors in both models of CMN explants and xenografts.

      Results

      Clinical and genetic characteristics

      Seventeen patients with a median age of 18 months and harboring lCMN/giant CMN were prospectively included: 10 L1 CMN, 6 L2 CMN, and 1 G1 CMN. Clinical characteristics are reported in Supplementary Table S1 online. NRAS and BRAF mutational status was ascertained using pyrosequencing and E-ice-COLD-PCR (see Supplementary Table S1). All samples displayed a single NRAS mutation on exon 3 with 12 NRAS Q61K mutations, 4 NRAS Q61R mutations, and 1 NRAS Q61L mutation.

      Overactivation of MAPK/Akt signaling pathways in NRAS-mutated lCMN

      To identify deregulated signaling pathways in lCMN, phosphorylation levels of NRAS downstream targets were compared between cultured nevocytes derived from 3 patients and 3 samples of NRAS wild-type normal human epidermal melanocytes using a human phospho-kinase array. The melanocytic lineage and the proliferative state of cultured cells were confirmed (see Supplementary Figure S1A online). The phospho-kinase arrays revealed an overall increase in the level of activated substrates in lCMN (Figure 1a). Akt isoforms displayed the most striking differences with a 13.25-fold increase (P = 0.0022) in phospho-Akt levels in lCMN. Phosphorylation levels of downstream targets of the MAPK pathway were also upregulated in lCMN, with 3.52- and 3.12-fold increases in phospho-ERK1 and phospho-ERK2 levels, respectively; 3.18- and 3.74-fold increases in phospho-p38α and phospho-p38γ levels, respectively; and a 3.82-fold increase in phospho–c-Jun N-terminal kinase 2 level (P < 0.05). Western blot analysis of lCMN cell cultures yielded similar results (Figure 1b and c). Immunohistochemical stainings on lCMN tissue sections confirmed previous results, displaying a higher staining intensity of phospho-MEK1/2(S221), phospho-ERK1/2(T202/Y204), and phospho-glycogen synthase kinase (GSK)-3β(S9) in both the epidermis and dermis of lCMN specimens when compared with normal skin (Figure 1d). This demonstrates an overall increase in the activation of the MAPK/PI3K-Akt signaling pathways downstream of NRAS in NRAS-mutated lCMN cells.
      Figure thumbnail gr1
      Figure 1NRAS-mutated human lCMN display an overactivation of MAPK/Akt signaling pathways. (a) Phosphokinase array analyzing expression of effector proteins of the MAPK/Akt signaling pathways in NHEM (n = 3 from 3 donors and lCMN cells (n = 3; 2 NRASQ61K + 1 NRAS Q61R). (b) Immunoblot analyses of effector proteins of the MAPK/Akt signaling pathways in NHEM (n = 3) and lCMN cells (n = 8; 7 NRASQ61K + 1 NRAS Q61R): ERK1/2, pERK1/2, Akt, pAkt, MEK, pMEK1/2, with (c) histogram of relative protein quantities. Vinculin was used as a loading control. (d) Immunohistochemical stainings of lCMN tissue specimens for pMEK1/2 (left panels), pERK1/2 (middle panels) and phospho-GSK-3β(S9) (right panels) in epidermis (upper panels) and dermis (lower panels) (n = 3; 1 NRASQ61K + 1 NRAS Q61R + 1 NRAS Q61L: pMEK1/2+, pERK1/2+ and phospho-GSK-3β+ cells were stained using acetate buffer, 3-amino-9-ethylcarbazole chromogen. *P < 0.05, **P < 0.01; represents differences between NHEM and lCMN cells in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; pAkt, phospo-Akt(S473); pERK1/2, phospho-ERK1/2(T202/Y204); pMEK1/2, phospho-MEK1/2(S221); NHEM, normal human epidermal melanocytes; SEM, standard error of mean. Scale bar = 100 μm.

      Decrease in lCMN cells clonogenic potential following MEK/Akt inhibition

      Our previous results prompted us to assess the consequences of a direct inhibition of MAPK and/or PI3K-Akt signaling pathways on nevocytes with clonogenic potential. Nevocytes from fresh lCMN cell suspensions were cultured as nevospheres, as previously described (
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      ,
      • Guégan S.
      • Kadlub N.
      • Picard A.
      • Rouillé T.
      • Charbel C.
      • Coulomb-L’Hermine A.
      • et al.
      Varying proliferative and clonogenic potential in NRAS-mutated congenital melanocytic nevi according to size.
      ), in the presence of binimetinib, a MEK inhibitor, and/or GSK690693, a pan-Akt inhibitor. Colonies’ diameter is an indicator of clonogenic potential (
      • Basu D.
      • Salgado C.M.
      • Bauer B.S.
      • Johnson D.
      • Rundell V.
      • Nikiforova M.
      • et al.
      Nevospheres from neurocutaneous melanocytosis cells show reduced viability when treated with specific inhibitors of NRAS signaling pathway.
      ;
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      ). On day 7, binimetinib induced a decrease in colonies’ diameter at all concentrations (vehicle: 55.10 ± 2.98 μm; 10 μM binimetinib: 38.03 ± 2.17 μm; P < 0.0001) (Figure 2a and b). On day 13, the colonies grown in the presence of the vehicle reached the maximum diameter, whereas the colonies cultured with binimetinib did not show an increase in diameter size (vehicle: 69.27 ± 3.47 μm; 10 μM binimetinib: 38.12 ± 1.58 μm; P < 0.0001) (Figure 2a and b). Inhibition of Akt induced a similar decrease in nevosphere diameter on day 7 and day 13 (Figure 2c and d). Finally, a combination of both inhibitors induced a reduction in diameters on both day 7 and day 13, with increasing concentrations of drugs inducing an increasing inhibitory effect on clonal proliferation (10 μM on day 7 and day 13: 38.73 ± 1.90 μm, P < 0.0001; 38.03 ± 2.61 μm, P < 0.0001, respectively) (Figure 2e and f). There were no significant differences in colonies’ diameters between cells cultured with sphere medium alone and vehicle (see Supplementary Figure S1b and c). The melanocytic lineage and proliferative state of colony-forming cells were confirmed (see Supplementary Figure S1d). Similar results were obtained with two additional inhibitors, MEK inhibitor PD0325901 and PI3Kα/δ inhibitor GDC-0941 (see Supplementary Figure S2 online). Altogether, these results demonstrate the effect of direct MEK/Akt inhibition on the proliferative potential of nevocytes initiating clonal proliferation.
      Figure thumbnail gr2
      Figure 2MEK/Akt inhibition impairs lCMN initiating cells proliferation in sphere-forming assays. (a, c, e) Photomicrographs and (b, d, f) histograms of lCMN colonies (n = 3; 2 NRASQ61K + 1 NRAS Q61R) cultured in sphere medium with vehicle or increasing concentrations of MEK (binimetinib)/Akt (GSK690693) inhibitors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; represents differences between vehicle and inhibitors in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/extracellular signal–regulated kinase kinase; SEM, standard error of mean. Scale bar = 100 μm.

      Cytostatic response of lCMN cells to MEK/Akt inhibition

      To further investigate the effects of MEK/Akt inhibition on nevocytes, inhibitors were tested on nevocytes in two-dimensional culture assays in M2 culture medium (Figure 3a). An in vitro proliferative MTT assay was also performed (Figure 3b). After 96 hours of culture, the MTT assay revealed a more significant effect of the inhibitors combination at all concentrations tested than that of binimetinib and GSK690693 alone (IC50 = 1.06 μM, 9.44 μM, and 8.73 μM, respectively), with only 21.84% of nevocytes viable at a concentration of 5 μM. A non–MAPK/PI3K-Akt inhibitor was used as a control; the Janus kinase inhibitor, tofacitinib, did not impair cell proliferation (see Supplementary Figure S3b online). To determine a potential cytotoxic effect of the inhibitors, a cytotoxicity assay was performed (Figure 3c). A rise in cell death was only noted with the combination of drugs at the highest concentrations, with significant cytotoxicity present only at 20 μM when compared with vehicle (25.45 ± 5.5% and 9.36 ± 1.41%, respectively, P = 0.04). Cleaved-caspase 3 immunocytostaining revealed the presence of apoptotic cells with the inhibitors combination (see Supplementary Figure S3a). Immunoblots were performed to assess the level of phosphorylated MEK1/2(S221), ERK1/2(T202/Y204), and GSK-3β(S9), an effector of the PI3K-Akt pathway (Figure 3d), with an impact of MEK and Akt inhibition on the levels of activated ERK1/2 and GSK-3β, respectively. Immunoblots of total proteins confirmed that expression levels were maintained with all inhibitors at all concentrations (see Supplementary Figure S3c). Altogether, these results demonstrate a potent and specific cytostatic effect of MEK/Akt inhibitors on nevocytes in vitro.
      Figure thumbnail gr3
      Figure 3MEK/Akt inhibition induces a cytostatic effect in lCMN cells. (a, b) Cell proliferation and (c) cytotoxicity assays in lCMN cells cultured with increasing concentrations of binimetinib/GSK690693 at 96 hours of culture (n = 3; 2 NRASQ61K + 1 NRAS Q61R). (d) Immunoblots analyses of downstream targets of the MAPK/PI3K-Akt signaling pathways (phospho-MEK1/2(S221), phospho-ERK1/2(T202/Y204), phospho-GSK-3β(S9)) in lCMN cells cultured with increasing concentrations of binimetinib/GSK690693 at 96 hours (n = 8; 7 NRASQ61K + 1 NRAS Q61R). Vinculin was used as a loading control. Cell viability and cytotoxicity were determined as a percentage of control. **P < 0.01; differences between vehicle and inhibitors in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol 3-kinase; SEM, standard error of mean. Scale bar = 100 μm.

      Decrease in nevocyte numbers in lCMN explants following MEK/Akt inhibition

      We set up a previously unreported ex vivo model of CMN by culturing standardized lCMN punch biopsy samples for 5 days (Figure 4a). Inhibitors did not alter explants histomorphology (Figure 4b and c, see Supplementary Figure S4a and b online). Immunohistochemical stainings of lCMN explants cultured with binimetinib or the inhibitors combination showed a significant decrease in phospho-ERK1/2 levels and a marked increase in phospho-MEK1/2 in the epidermis and dermis (Figure 4d, see Supplementary Figure S4c). Treatment with GSK690693 induced a decrease in phospho-GSK-3β levels. Similar staining modifications were not obtained in the epidermis and dermis of normal skin explants (see Supplementary Figure S5 online).
      Figure thumbnail gr4
      Figure 4MEK/Akt inhibition induces modifications in lCMN explants histology. (a) Hematoxylin and eosin stainings of lCMN tissue specimens and explants on day 0 and day 5 of ex vivo culture. Dotted lines delineate neoepidermal tongues. (b) Hematoxylin and eosin stainings of day 5 lCMN explants cultured with or without binimetinib/GSK690693, including close-up views of the epidermis (left panels) and dermis (right panels). (c) Histogram of epidermal thickness of day 5 lCMN explants cultured with or without binimetinib/GSK690693 (n = 3; 2 NRASQ61K + 1 NRAS Q61R). (d) Immunohistochemical stainings for phospho-MEK1/2(S221) (upper panels), phospho-ERK1/2(T202/Y204) (middle panels), and phospho-GSK-3β(S9) (lower panels) of lCMN explants cultured with or without binimetinib/GSK690693 (n = 3; 2 NRASQ61K + 1 NRAS Q61L). Phospho-MEK1/2+, phospho-ERK1/2+, and phospho-GSK-3β+ cells were stained using AEC chromogen. *represents differences between vehicle and inhibitor-treated explants. Akt, protein kinase B; ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase /ERK kinase. Scale bar = 100 μm.
      To characterize the cellular composition of lCMN explants, the percentage of MelanA+ cells was assessed (Figure 5a–c, see Supplementary Figure S6a online). Immunofluorescent stainings revealed a significant decrease in the number of MelanA+ cells in the dermis of explants cultured with 25 μM inhibitors alone and in combination: vehicle 25.75 ± 1.69% of total cells, binimetinib 11.91 ± 1.06% (P < 0.0001), GSK690693 12.01 ± 1.50% (P < 0.0001), and combination 12.19 ± 1.36% (P < 0.0001). A similar decrease was evidenced in the epidermis (Figure 5b and c). Comparable results were obtained at 50 μM with all inhibitors (Figure 5b and c).
      Figure thumbnail gr5
      Figure 5MEK/Akt inhibition decreases nevocyte numbers in lCMN explants. Immunofluorescent stainings and quantification of lCMN explants cultured with or without binimetinib/GSK690693: (a–c) MelanA staining and (d–f) Sox10/Ki67 double staining in epidermis and dermis (n = 3; 2 NRASQ61K + 1 NRAS Q61L). MelanA+ and Sox10+ cells were stained using Alexa488-conjugated antibodies, and Ki67+ cells were stained using Cy3-conjugated antibodies. Histograms show the number of positive cells per total cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; represents differences between vehicle and inhibitor-treated explants in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/extracellular signal–regulated kinase kinase; ND, not detected; SEM, standard error of mean. Scale bar = 100 μm.
      Immunofluorescent stainings with Sox10/Ki67 were also performed (Figure 5d–f, see Supplementary Figure S6b). Analysis revealed a significant decrease in the number of Sox10+ cells in the dermis of lCMN explants cultured with 25 μM inhibitors: vehicle 15.72 ± 2.06% of total cells, binimetinib 8.26 ± 1.37% (P = 0.0266), GSK690693 3.31 ± 0.63% (P < 0.0001), and combination 4.61 ± 0.83% (P <0.0001), as well as in the epidermis (Figure 5e and f). Comparable results were obtained with 50 μM inhibitors (Figure 5e and f).
      The epidermis of normal skin explants did not display a similar reduction in MelanA+ and Sox10+ cell numbers (see Supplementary Figure S7 online).
      Quantification of Ki67+ cells showed a decrease in proliferating cells in the epidermis and dermis of lCMN explants; there were no Ki67+ cells in explants cultured with binimetinib alone and combination at both concentrations (Figure 5d–f, see Supplementary Figure S6b). GSK690693 treatment induced an increase in the percentage of Ki67+ cells (Figure 5d–f, see Supplementary Figure S6b). We could not detect double-positive Sox10+/Ki67+ cells in any lCMN explants (Figure 5d, see Supplementary Figure S6b). A double keratin14 (K14)/Ki67 immunofluorescent staining was performed and revealed that all proliferating cells were of the keratinocyte lineage (see Supplementary Figure S6c). Normal skin explants yielded similar results (see Supplementary Figure S7). Cleaved-caspase 3 immunofluorescent staining revealed the presence of apoptotic cells in the dermis of lCMN explants treated with inhibitors (see Supplementary Figure S6d).
      In the course of the culture, the development of a neoepidermal tongue was impaired by the use of inhibitors (see Supplementary Figure S8a and b online). MelanA+ and Sox10+ cells were present in the neoepidermal tongue of explants except when cultured with inhibitors (see Supplementary Figure S8c and d).
      In conclusion, MEK/Akt inhibition dramatically reduces nevocyte numbers in our ex vivo model of lCMN explant, whereas melanocytes in normal skin explants are not affected in a similar way.

      Decrease in nevocyte and proliferative cell numbers in lCMN xenografts following MEK/Akt inhibition

      To further investigate MEK and/or Akt inhibition for lCMN treatment, full-thickness lCMN xenografts were performed on the back of immunocompromised Rag2–/– mice (Figure 6a). Two months postgrafting, inhibitors were injected daily intradermally within xenografts during 8 days (Figure 6b). Xenografts were then harvested 1 day (day 1 xenografts) or 30 days (day 30 xenografts) after the end of intradermal injections. In hematoxylin and eosin stainings, xenografts showed a histomorphological structure very similar to that of the original CMN with melanocytes extending from the epidermis to the deep dermis (Figure 6c). The use of inhibitors did not alter this structure in day 1 xenografts (Figure 6c, see Supplementary Figure S9a online). Fontana-Masson stainings were also performed to assess the pigment content in day 1 xenografts (see Supplementary Figure S9b). In the case of markedly pigmented nevi, inhibitors induced a decrease in the number of nevocytes and melanin deposits within nevocytes in the epidermis and dermis of treated xenografts and a significant increase in melanophages in the dermis. MelanA immunofluorescent stainings revealed a decrease in the percentage of positive cells in day 1 xenografts treated with inhibitors alone and in combination, in both epidermis (vehicle: 6.56 ± 0.50% of total cells; binimetinib: 4.34 ± 0.49%, P = 0.0013; GSK690693: 1.66 ± 0.35%, P 0.0001; combination: 1.62 ± 0.27%, P <0.0001) and dermis (vehicle: 31.07 ± 2.61% of total cells; binimetinib: 11.20 ± 1.57%, P < 0.0001; GSK690693: 7.49 ± 1.76%, P < 0.0001; combination: 1.78 ± 0.39%, P < 0.0001) (Figure 6d). A comparable effect was detected with Sox10/Ki67 double staining in day 1 xenografts in both epidermis (vehicle: 6.42 ± 0.49% of total cells; binimetinib: 3.55 ± 0.54%, P < 0.0001; GSK690693: 1.55 ± 0.33%, P < 0.0001; combination: 1.67 ± 0.38%, P < 0.0001) and dermis (vehicle: 41.08 ± 2.97% of total cells; binimetinib: 16.08 ± 2.73%, P < 0.0001; GSK690693: 6.49 ± 2.19%, P < 0.0001; combination: 3.56 ± 0.99%, P < 0.0001) (Figure 6e). Similar results were obtained in day 30 xenografts (see Supplementary Figure S10a online) with a significant decrease both in MelanA+ and Sox10+ cells after combination therapy when compared with vehicle (see Supplementary Figure S10b and c). Ki67 stainings showed a decrease in proliferating cells in day 1 xenografts treated with inhibitors alone or in combination in both the epidermis and dermis (Figure 6e). Fewer Sox10+/Ki67+ cells were present in the epidermis of the day 1 xenografts treated with binimetinib or a combination of inhibitors. Sox10+/Ki67+ cells were neither detected in the epidermis of GSK690693 treated xenografts nor in the dermis of any inhibitor-treated xenografts (Figure 6f). Immunohistochemical stainings for phospho-ERK1/2(T202/Y204) and phospho-GSK-3β(S9) showed a decrease in staining intensity when xenografts were treated with binimetinib and GSK690693, respectively, but no change in phospho-MEK1/2(S221) (Figure 6g, see Supplementary Figure S10d). K14/Ki67 immunofluorescent staining highlighted the keratinocyte lineage of most proliferating cells found in the epidermis (see Supplementary Figure S11a online). Cleaved-caspase 3 immunofluorescent staining did not reveal the presence of apoptotic cells in inhibitor-treated xenografts (see Supplementary Figure S11b and c). Human mitochondria immunofluorescent stainings confirmed that xenografts were exclusively constituted of human cells (see Supplementary Figure S11d). The same experiments were conducted on normal skin xenografts (see Supplementary Figure S12 online). As with lCMN xenografts, the treatment did not induce modifications of the histomorphological structure of the skin, and immunohistochemical stainings for phospho-MEK1/2 and phospho-ERK1/2 yielded similar results (see Supplementary Figure S12a and b). Quantification of MelanA+, Sox10+, and Ki67+ cells showed no decrease in the number of melanocytes in the epidermis of normal skin xenografts, and K14+/Ki67+ immunofluorescent staining confirmed the keratinocyte lineage of proliferating cells (see Supplementary Figure S12c). Daily treatment did not seem to induce harmful side effects; mice behavior remained normal, and hematoxylin and eosin stainings did not show any alterations in the histological structure of the main organs susceptible to targeted therapy toxicity (see Supplementary Figure S13 online). These results demonstrate a successful decrease in nevocyte numbers in day 1 lCMN xenografts following MEK/Akt inhibition with a persisting effect in day 30 xenografts, while melanocytes in normal skin are not affected.
      Figure thumbnail gr6
      Figure 6MEK/Akt inhibition decreases nevocyte and proliferative cell numbers in day 1 lCMN xenografts. (a) Photograph of lCMN tissue xenograft on immunocompromised Rag2–/– mouse 60 days after xenografting. (b) After lCMN specimens xenografting, a 2-month recovery period was allowed to ensure complete engraftment. Intradermal injections were performed daily for 8 days and xenografts harvested for further analysis on day 1 postinjection. (c) Hematoxylin and eosin staining of lCMN tissue specimen and corresponding xenografts injected with vehicle or inhibitors. (d–f) Immunofluorescent stainings and quantification of lCMN tissue xenografts injected with or without binimetinib/GSK690693: MelanA staining (d) and Sox10/Ki67 double staining (e, f) in epidermis and dermis. MelanA+ and Sox10+ cells were stained using Alexa488-conjugated antibodies; Ki67+ cells were stained using Cy3-conjugated antibodies. (g) Immunohistochemical stainings of lCMN xenografts injected with vehicle or inhibitors for phospho-MEK1/2(S221) (upper panels), phospho-ERK1/2(T202/Y204) (middle panels), and phospho-GSK-3β(S9) (lower panels). Phospho-MEK1/2+, phospho-ERK1/2+, and phospho-GSK-3β+ cells were stained using AEC chromogen. Histograms show the number of positive cells per total cells. n = 3; 1 NRASQ61K + 1 NRAS Q61R + 1 NRAS Q61L. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; represents differences between vehicle and inhibitor-treated xenografts in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/ERK kinase; ND, not detected; SEM, standard error of mean. Scale bar = 100 μm.

      Discussion

      We report MEK1/2, ERK1/2, and Akt overactivation in CMN. Accordingly, we tested the efficiency of inhibitors targeting MAPK and Akt signaling cascades. MEK and/or Akt inhibition induced a decrease in nevocyte clonogenic potential through a cytostatic effect. In a previously unreported model of CMN explants, these inhibitors decreased the nevocytic population, particularly when inhibitors were combined. Finally, a model of CMN xenograft preserving the histomorphology of the original nevus allowed the assessment of a local intradermal treatment with these same drugs. Inhibitor-treated CMN xenografts were characterized by an important reduction in the number of nevocytes in both epidermis and dermis with a more pronounced effect on samples treated with both MEK and Akt inhibitors. This effect persisted 30 days after the end of treatment. As phospho-ERK1/2 seems to be the most strongly affected signaling mediator in lCMN, the use of ERK inhibitors would also be of interest.
      Our in vitro studies showed that clonogenic cell culture of CMN nevocytes may be used as a valuable tool for the evaluation of inhibitory molecules of potential use for CMN treatment.Furthermore, a comparable clonogenic potential of neurocutaneous melanocytosis-derived cells was reported by others (
      • Basu D.
      • Salgado C.M.
      • Bauer B.S.
      • Johnson D.
      • Rundell V.
      • Nikiforova M.
      • et al.
      Nevospheres from neurocutaneous melanocytosis cells show reduced viability when treated with specific inhibitors of NRAS signaling pathway.
      ). In accordance, MEK and PI3K/mTOR inhibition led to a decrease in spheroids size in one patient’s culture.
      Our ex vivo model of CMN explants proved relevant for assessing drug efficiency in CMN; it allowed the evaluation of cell numbers, cell proliferation, and characterization in all compartments of the tumor but also of cellular dynamics, as cells retained their ability to proliferate and migrate (
      • Meier N.T.
      • Haslam I.S.
      • Pattwell D.M.
      • Zhang G.Y.
      • Emelianov V.
      • Paredes R.
      • et al.
      Thyrotropin-releasing hormone (TRH) promotes wound re-epithelialisation in frog and human skin.
      ,
      • Nguyen V.T.
      • Farman N.
      • Maubec E.
      • Nassar D.
      • Desposito D.
      • Waeckel L.
      • et al.
      Re-epithelialization of pathological cutaneous wounds is improved by local mineralocorticoid receptor antagonism.
      ,
      • Le Poole I.C.
      • Van den Wijngaard R.M.
      • Westerhof W.
      • Dormans J.A.
      • Van den Berg F.M.
      • Verkruisen R.P.
      • et al.
      Organotypic culture of human skin to study melanocyte migration.
      ). Moreover, it required limited amounts of patients’ tissues. Several studies have highlighted the use of ex vivo skin culture as an adequate tool for wound healing studies and for the screening of various molecules (
      • Meier N.T.
      • Haslam I.S.
      • Pattwell D.M.
      • Zhang G.Y.
      • Emelianov V.
      • Paredes R.
      • et al.
      Thyrotropin-releasing hormone (TRH) promotes wound re-epithelialisation in frog and human skin.
      ,
      • Nguyen V.T.
      • Farman N.
      • Maubec E.
      • Nassar D.
      • Desposito D.
      • Waeckel L.
      • et al.
      Re-epithelialization of pathological cutaneous wounds is improved by local mineralocorticoid receptor antagonism.
      ,
      • Rizzo A.E.
      • Beckett L.A.
      • Baier B.S.
      • Isseroff R.R.
      The linear excisional wound: an improved model for human ex vivo wound epithelialization studies.
      ,
      • Xu W.
      • Jong Hong S.
      • Jia S.
      • Zhao Y.
      • Galiano R.D.
      • Mustoe T.A.
      Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.
      ). Several murine models involving NRAS mutations are available for CMN study (
      • Ackermann J.
      • Frutschi M.
      • Kaloulis K.
      • McKee T.
      • Trumpp A.
      • Beermann F.
      Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background.
      ,
      • Pedersen M.
      • Küsters-Vandevelde H.V.N.
      • Viros A.
      • Groenen P.J.T.A.
      • Sanchez-Laorden B.
      • Gilhuis J.H.
      • et al.
      Primary melanoma of the CNS in children is driven by congenital expression of oncogenic NRAS in melanocytes.
      ,
      • Shakhova O.
      • Zingg D.
      • Schaefer S.M.
      • Hari L.
      • Civenni G.
      • Blunschi J.
      • et al.
      Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma.
      ). Though the murine models display features similar to the human nevi, their value for therapeutic assessment is limited, as murine skin structure differs from human skin. We consider that CMN xenografts are the preclinical proof of concept for medical treatment in this condition. These grafts allowed the study of primary human tissue and the screening of molecules injected in situ. This model could also be of use for the study of CMN progression, as CMN is considered a growth arrested if not senescent tumor (
      • Michaloglou C.
      • Vredeveld L.C.W.
      • Soengas M.S.
      • Denoyelle C.
      • Kuilman T.
      • van der Horst C.M.A.M.
      • et al.
      BRAFE600-associated senescence-like cell cycle arrest of human naevi.
      ,
      • Tran S.L.
      • Haferkamp S.
      • Scurr L.L.
      • Gowrishankar K.
      • Becker T.M.
      • Desilva C.
      • et al.
      Absence of distinguishing senescence traits in human melanocytic nevi.
      ).
      The use of small inhibitory molecules has shown promising results in cancer treatment, including NRAS-mutated melanoma. Posch and colleagues (2013) reported that inhibition of either MAPK or PI3K-Akt pathways in several melanoma cell lines decreased cell viability in vitro, and double-targeting led to cell death in vitro and tumor regression in xenografted immunocompromised mice. In a phase 2 study, MEK inhibition with binimetinib showed clinical relevance for the treatment of NRAS-mutated metastatic melanoma (
      • Ascierto P.A.
      • Schadendorf D.
      • Berking C.
      • Agarwala S.S.
      • van Herpen C.M.
      • Queirolo P.
      • et al.
      MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study.
      ). Several cases of MEK inhibition in patients with neurocutaneous melanocytosis and central nervous system melanoma, conditions associated with CMN, have also been reported. Küsters-Vandevelde and colleagues (2014) described the case of a 13-year-old patient suffering from NRAS-mutated neurocutaneous melanocytosis and treated with binimetinib. However, as opposed to our study, the treatment was systemic. The use of targeted inhibitors as local treatment should limit the side effects described with their systemic use.
      In conclusion, we describe 2 preclinical models allowing drug screening for CMN treatment. The preclinical model of intradermal drug injections in patient-derived CMN xenografts could be used to assess the efficiency of local therapy in a patient-specific manner. The development of a local intradermal treatment is of importance in children as it should cause less adverse events than a systemic targeted therapy. Various vehicles, such as nanoparticle-bound or pegylated inhibitors, could eventually allow an extended duration of action of the drugs, thus allowing fewer patient injections. This treatment could be used both before and/or following surgery to prevent CMN cellular growth, thus reducing the physical trauma caused by multiple surgical procedures (
      • Bellier-Waast F.
      • Perrot P.
      • Duteille F.
      • Stalder J.F.
      • Barbarot S.
      • Pannier M.
      [Surgical treatment for giant congenital nevi: what are the psychosocial consequences for the child and family?].
      ). It could also affect quiescent dermal nevus cells, thus preventing the risk of malignant transformation as these cells may constitute the reservoir responsible for melanoma emergence through accumulation of mutations (
      • Grichnik J.M.
      • Ross A.L.
      • Schneider S.L.
      • Sanchez M.I.
      • Eller M.S.
      • Hatzistergos K.E.
      How, and from which cell sources, do nevi really develop?.
      ,
      • Shain A.H.
      • Yeh I.
      • Kovalyshyn I.
      • Sriharan A.
      • Talevich E.
      • Gagnon A.
      • et al.
      The genetic evolution of melanoma from precursor lesions.
      ). Finally, reducing CMN size may offer beneficial effects on socioaesthetic-related issues as major psychosocial impact is reported in patients harboring large CMN and their families.

      Materials and Methods

      Details can be found in Supplementary Materials and Methods online.

      Study approval

      The study was approved by the institutional independent ethics committee (Comité de Protection des Personnes Ile-de-France V) and complied with the Declaration of Helsinki Principles. The patients’ guardians provided written informed consent before their participation. CMN were obtained from the plastic surgery department of Necker-Enfants-Malades hospital. Clinical phenotyping of patients was performed by SG and NK. Classification was done using the projected adult size of the largest lesion.

      In vitro assays

      Fresh lCMN tissue specimens cell suspensions were obtained as previously reported (
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      ,
      • Guégan S.
      • Kadlub N.
      • Picard A.
      • Rouillé T.
      • Charbel C.
      • Coulomb-L’Hermine A.
      • et al.
      Varying proliferative and clonogenic potential in NRAS-mutated congenital melanocytic nevi according to size.
      ). Normal human epidermal melanocytes from juvenile patients (PromoCell, Heidelberg, Germany) and fresh nevocytic cell suspensions derived from patients were cultured in either spheres or M2 complete medium (PromoCell) and analyzed using dot blots/western blots. Proliferation and cellular death of nevocytes were assessed using Cell Titer 96 Non-Radioactive Cell Proliferation Assay and CytoTox 96 Non-Radioactive Cytotoxicity Assay, respectively (Promega, Madison, WI).

      Ex vivo assays

      Human lCMN and normal skin explants were obtained by performing 3 mm skin punch biopsies after mechanic removal of the hypodermis. Explants were placed in organotypic culture for 5 days in DMEM/F12 supplemented with epidermal growth factor (100 ng/ml), basic fibroblast growth factor (100 ng/ml), 10% decomplemented fetal calf serum, and insulin (5 μg/ml). Explants were cultured with or without drugs. After 5 days of incubation, explants were harvested, washed with phosphate buffered saline, fixed in 4% paraformaldehyde and paraffin-embedded. Sections were stained with hematoxylin and eosin (Thermo Fisher Scientific, Waltham, MA).

      In vivo assays

      Animal experiments were performed according to experimental protocols following European Community Council guidelines and approved by our Institutional Animal Care and Use Committee. Full-thickness lCMN and normal skin tissues of 1 cm2 were xenografted on the back skin of Rag2–/– mice. Intradermal injections of 100 μl of drugs diluted in 45% polyethylen glycol 300 with 5% DMSO were then performed on xenografts every 24 hours for 8 days. Xenografts were harvested on the 1st day or the 30th day after the end of injections, fixed in 4% paraformaldehyde, and embedded in paraffin.

      Data availability statement

      There are no large datasets (such as gene expression arrays, single-nucleotide polymorphism arrays, proteomic datasets, high-throughput sequencing, and genome-wide association study data) linked to this publication.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We are grateful to all the patients and families who participated in this study. We thank Dany Nassar for helpful discussions. We thank Maria Sbeih, Aleksandra Savina, Aude Fert, Michèle Oster, Romain Morichon and Imaging Platform UMS30 LUMIC, Tatiana Ledent, and the Saint-Antoine Research Center Animal Facility for their technical support. This work was funded by the Société Française de Dermatologie and AREMPH. TR received support from the French Ministry of Education and the Fondation pour la Recherche Médicale (FDT20170436873).

      Author Contributions

      Conceptualization: SA, RHF, SG; Formal Analysis: TR, SA, NK, AH-K, PM, RHF, SG; Funding Acquisition: SA, RHF, SG; Investigation: TR, NK, SF, AH-K, AD, MH, PM, AP, RHF, SG; Project Administration: SA, RHF, SG; Supervision: SA, RHF, SG; Validation: TR, AH-K, RHF, SG; Visualization: TR, RHF, SG; Writing - Original Draft Preparation: TR, RHF, SG; Writing - Review and Editing: TR, SA, RHF, SG

      Supplementary Materials and Methods

      Genotyping

      DNA extraction, standard real-time PCR, e-ice-COLD-PCR, and pyrosequencing were performed as previously described (
      • Charbel C.
      • Fontaine R.H.
      • Malouf G.G.
      • Picard A.
      • Kadlub N.
      • El-Murr N.
      • et al.
      NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi.
      ;
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      ).

      Reagents and antibodies

      All drugs used in this study were obtained from Selleck Chemicals (Houston, TX), diluted in DMSO (Sigma-Aldrich, Saint Louis, MO), and used at various concentrations, ranging from 10 nM to 20 μM for in vitro assays. Binimetinib and GSK690693 were both used at 25 μM and 50 μM for ex vivo assays, and at 6 μM and 38 μM for in vivo assays (daily injections during 8 days within xenografts). Antibodies used are listed in Supplementary Materials and Methods online.

      Dot blots/western blots

      Normal human epidermal melanocytes from juvenile patients (PromoCell) and fresh nevocytic cell suspensions derived from patients were cultured in M2 complete medium (PromoCell). For dot blot analysis (using human phospho-kinase array, R&D Systems, Minneapolis, MN), cells were harvested and proteins extracted and revealed according to the manufacturer protocol. For western blots analysis, cells were washed using phosphate buffer saline and harvested and cytosolic proteins extracted with radioimuunoprecipitation buffer (Tris-HCl 50 mM pH 7.5, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 5 mM EDTA) supplemented with phosphatase and protease inhibitors (Roche, Basel, Switzerland). Proteins (20 μg) were separated using SDS-PAGE, transferred on a nitrocellulose membrane, and blocked for 2 hours in blocking buffer (tris-buffered saline with 0.1% Tween 20 [TBST] and 5% dry milk). Membranes were incubated overnight at 4 °C with primary antibodies diluted at 1:1000 in TBST with 5% BSA, followed by 1-hour incubation with peroxidase-conjugated secondary antibody diluted at 1:5000 in blocking buffer, and revealed using chemoluminescence.

      Antibodies

      Antibodies that were used included anti-Vinculin, anti-phospho-MEK1/2 (Ser221), anti-phospho-p44/42 (ERK1/2) (Thr202/Tyr204), anti-phospho-Akt (Ser473), anti-phospho-GSK-3ß (Ser9), anti-MEK1/2, anti-p44/42 (ERK1/2), anti-Akt, anti-GSK-3ß, anti-cleaved-caspase 3, and anti-Sox10 obtained from Cell Signaling Technology (Danvers, MA), anti-MelanA obtained from Abcam (Cambridge, UK), anti-Ki67 obtained from DAKO (Santa Clara, CA), K14 obtained from BioLegend (San Diego, CA) and anti-human-mitochondria obtained from Millipore (Temecula, CA).

      Sphere assay

      Fresh nevocytic cell suspensions deriving from patients were cultured as previously described (
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      ), with or without inhibitors. DMSO and sphere medium were used as controls. Nevosphere diameters were measured between 5 days and 13 days of culture.

      Proliferation/Cytotoxicity assays

      Proliferation and cellular death of nevocytes derived from patients after drug treatment was assessed using Cell Titer 96 Non-Radioactive Cell Proliferation Assay and CytoTox 96 Non-Radioactive Cytotoxicity Assay, respectively (Promega). Briefly, large congenital melanocytic nevi (lCMN) cell suspensions were seeded in M2 complete medium at a density of 5,000 cells per well for the proliferation assay and 2,500 cells per well for the cytotoxicity assay. Proliferation and cytotoxicity were measured at 96 hours according to the manufacturer protocol.

      Immunostaining

      Formalin–fixed paraffin-embedded lCMN, normal skin, explants, and xenografts sections (5 μm) were labeled with the following antibodies: mouse anti-human phospho-MEK1/2 (dilution 1:50), mouse anti-human phospho-p44/42 (ERK1/2) (dilution 1:50), mouse anti-human KI67 (dilution 1:100), mouse anti-human MelanA (dilution 1:100), rabbit anti-human Sox10 (dilution 1:100), rabbit anti-human K14 (dilution 1:1000), and rabbit anti-human cleaved-caspase 3. Antigen retrieval was performed by incubating the slides with citrate buffer (pH = 6) or EDTA buffer (pH = 9) at 98 °C for 20 minutes. For phospho-MEK1/2 and phospho-p44/42 (ERK1/2) stainings, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Sections were washed with tris-buffered saline (137 mM NaCl and 20 mM Tris) with 0.1% TBST and blocked for 2 hours with TBST with 5% normal goat serum. The sections were incubated with the different primary antibodies diluted in blocking buffer overnight at 4 °C for intracellular staining. Staining was achieved using appropriate biotinylated goat anti-mouse secondary antibodies (Vector Laboratories, Burlingame, CA) and Avidine Biotine Complex (Vector Laboratories). Bound antibodies were visualized using aminoethylcarbazol (Vector Laboratories) as chromogen. Fluoride sodium (0.1 mM) was added to all buffers and incubation solutions.
      For MelanA, Sox10, Ki67, K14, and cleaved-caspase 3 immunofluorescence, slides were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. Sections were washed with TBST followed by secondary antibody goat anti-mouse Alexa 488 (Life Technologies, Carlsbad, CA) in a dilution of 1:1000, goat anti-mouse Alexa 546 (Life Technologies) in a dilution of 1:1000, or goat anti-rabbit Alexa 488 (Life Technologies) in a dilution of 1:1000 for 45 minutes at room temperature. Slides were also incubated with DAPI (Sigma-Aldrich) for 5 minutes for nuclei staining and coverslipped using Fluoromount G (Southern Biotech, Birmingham, AL). Negative controls were performed in parallel with the samples substituting the primary antibody with the equivalent isotype. Photomicrographs were obtained under either ×10 or ×20 objective using Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan) and Olympus IX83 microscope (Olympus, Tokyo, Japan). Using Image J software (NIH, Bethesda, MD), positive cells were counted in 104 μm2 randomized surface squares in both the epidermis and dermis of each specimen. In each area, the number of positive cells was counted and reported to the total number of DAPI+ cells. Thus, results are expressed as a percentage of positive cells for each marker analyzed per total cells per 104 μm2 surface areas, both in the epidermis and dermis. For lCMN tissue explants, the entire neoepidermal tongue was counted. Quantifications were done on various parts of the lCMN and normal skin and always in areas within the lesions, usually in the center; these lesions were dissected in a gridded fashion and 3 different regions were analyzed. For each different region of the lesions, 3 different areas of 104 μm2 were counted.
      For immunocytochemistry, stainings were performed as previously described
      • Charbel C.
      • Fontaine R.H.
      • Kadlub N.
      • Coulomb-L’Hermine A.
      • Rouillé T.
      How-Kit A, et al. Clonogenic cell subpopulations maintain congenital melanocytic nevi.
      . Adherent monoloayers and sphere colonies were stained with anti-MelanA (1:100), anti-Sox10 (1:100), anti-Ki67 (1:100), and anti-cleaved-caspase 3 (1:100) antibodies.

      Epidermal thickness

      Epidermal thickness of lCMN and normal skin explants and xenografts were measured on various parts of the tissue using Image J software. Three different tissue sections were analyzed per sample; on each section, 3 different areas were measured.

      Statistics

      The data were analyzed using Mann-Whitney U test. For all results, n indicates the number of independent experiments performed. In all histograms, asterisks correspond to *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
      Figure thumbnail fx1
      Supplementary Figure S1In vitro analysis of lCMN cells. (a) Immunocytofluorescent staining of cultured lCMN cells stained with MelanA (left panel) and Sox10/Ki67 double staining (right panel). (b) Photomicrographs, (c) histograms, and (d) immunostainings of lCMN colonies (n = 3) cultured in sphere medium with or without vehicle. MelanA+ and Sox10+ cells were stained using Alexa488-conjugated antibodies. Ki67+ cells were stained using Cy3-conjugated antibodies. Error bars: mean ± SEM. lCMN, large congenital melanocytic nevi; SEM, standard error of mean. Scale bar = 100 μm.
      Figure thumbnail fx2
      Supplementary Figure S2MEK/Akt inhibition impairs lCMN initiating cells proliferation in sphere-forming assays. Histograms of lCMN colonies (n = 3; 1 NRAS Q61R + 2 NRAS Q61K) cultured in sphere medium with vehicle or increasing concentrations of MEK (PD0325901), PI3K (GDC0941), or Akt (GSK690693) inhibitors. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; represents differences between vehicle and inhibitors in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/extracellular signal–regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; SEM, standard error of mean.
      Figure thumbnail fx3
      Supplementary Figure S3In vitro and signaling pathway analysis of lCMN cells. (a) Immunocytofluorescent staining of cultured lCMN cells stained with cleaved-caspase 3 (Alexa488-conjugated secondary antibodies). (b) Cell proliferation assays with lCMN cells (n = 2; 2 NRAS Q61K) cultured with increasing concentrations of tofacitinib and comparison with the vehicle at 96 hours of culture. (c) Immunoblots of total proteins of the MAPK/Akt signaling pathways in lCMN cells cultured with increasing concentrations of binimetinib/GSK690693 as compared with vehicle. Vinculin was used as loading control (n = 3; 3 NRAS Q61K). Error bars: mean ± SEM. Akt, protein kinase B; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MAPK, mitogen-activated protein kinase; SEM, standard error of mean. Scale bar = 100 μm.
      Figure thumbnail fx4
      Supplementary Figure S4Ex vivo analysis of lCMN tissue explants cultured with 50 μM inhibitors. (a) Hematoxylin and eosin stainings of lCMN tissue explants cultured for 5 days with or without inhibitors. (b) Measurement of epidermal thickness of lCMN tissue explants cultured for 5 days with or without inhibitors at 50 μM (n = 3; 2 NRASQ61K + 1 NRAS Q61R). (c) Immunohistochemical stainings of lCMN tissue explants (n = 3; 2 NRASQ61K + 1 NRAS Q61L) cultured with or without inhibitors for phospho-MEK1/2(S221) (upper panels) and phospho-ERK1/2 (T202/Y204) (lower panels). Phospho-MEK1/2 and phospho-ERK1/2 positive cells were stained using AEC chromogen. Dotted lines delineate neoepidermal tongue. Akt, protein kinase B; ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/ERK kinase. Scale bar = 100 μm.
      Figure thumbnail fx5
      Supplementary Figure S5Ex vivo analysis of normal skin tissue explants cultured with inhibitors. (a) Hematoxylin and eosin (upper panels), phospho-MEK1/2 (S221), phospho-ERK1/2 (T202/Y204), and phospho-GSK-3β (S9; lower panels) immunohistochemical stainings of normal skin explants cultured for 5 days with or without inhibitors at 25 μM. Phospho-MEK1/2, phospho-ERK1/2, and phospho-GSK-3β positive cells were stained using AEC chromogen. (b) Measurement of epidermal thickness of normal skin tissue explants cultured for 5 days with or without inhibitors at 25 μM and 50 μM. n = 3 from 3 donors. ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; MEK, mitogen-activated protein kinase/ERK kinase. Scale bar = 100 μm.
      Figure thumbnail fx6
      Supplementary Figure S6Ex vivo cellular characterization of lCMN tissue explants cultured with 50 μM inhibitors. (a–c) Immunofluorescent stainings of lCMN tissue explants with or without inhibitors (n = 3; 2 NRASQ61K + 1 NRAS Q61L): (a) MelanA, (b) Sox10/Ki67 (c) K14/Ki67 in epidermis, dermis, and neoepidermal tongue. (d) Cleaved-caspase 3 immunofluorescent stainings and histograms in epidermis and dermis. MelanA+, Sox10+, K14+, and cleaved-caspase 3+ cells were stained using Alexa488-conjugated antibodies. Ki67+ cells were stained using Cy3-conjugated antibodies. Dotted lines delineate neoepidermal tongue. *P < 0.05, **P < 0.001, ***P < 0.001; Mann-Whitney U test. lCMN, large congenital melanocytic nevi. Scale bar = 100 μm.
      Figure thumbnail fx7
      Supplementary Figure S7Ex vivo analysis of normal skin tissue explants cultured with inhibitors. Immunofluorescent stainings and histograms of normal skin explants (n = 3 from 3 donors) cultured with or without inhibitors for MelanA (left panels), Sox10/Ki67 (middle panels), K14/Ki67 (right panels). MelanA+, Sox10+, and K14+ cells were stained using Alexa488-conjugated antibodies. Ki67+ cells were stained using Cy3-conjugated antibodies. *P < 0.05, **P < 0.01, ***P < 0.001; represents differences between control and inhibitor-treated explant in Mann-Whitney U test. Error bars: mean ± SEM. ND, not detected. Scale bar = 100 μm.
      Figure thumbnail fx8
      Supplementary Figure S8MEK/Akt inhibition induces modifications in the neoepidermal tongue of lCMN explants. Day 5 lCMN explants cultured with or without inhibitors. (a) Hematoxylin and eosin stainings with close-up views of neoepidermal tongues. (b) Histogram of neoepidermal tongue length, expressed as a percentage of lCMN explant total length (n = 3, 2 NRASQ61K + 1 NRAS Q61L). (c, d) Immunohistochemical stainings and quantification of lCMN explants stained with (c) MelanA and (d) Sox10 in epidermis, dermis, and neoepidermal tongue (n = 3, 2 NRASQ61K + 1 NRAS Q61L), using Alexa488-conjugated antibodies. Histograms show the number of positive cells per total cells. Dotted lines delineate neoepidermal tongues. *P < 0.05, **P < 0.001, ****P < 0.0001; represents differences between control and inhibitor-treated explants in Mann-Whitney U test. Error bars: mean ± SEM. Akt, protein kinase B; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/ extracellular signal–regulated kinase kinase; ND, not detected; SEM, standard error of mean. Scale bar = 100 μm.
      Figure thumbnail fx9
      Supplementary Figure S9In vivo characterization of day 1 lCMN xenografts. (a) Measurement of epidermal thickness of day 1 lCMN tissue xenografts injected with or without inhibitors compared with the original CMN. (b) Fontana-Masson stainings of original lCMN and lCMN xenografts injected with or without inhibitors and harvested on day 1 postinjections. n = 3; 1 NRASQ61K + 1 NRAS Q61R + 1 NRASQ61L. CMN, congenital melanocytic nevi; lCMN, large congenital melanocytic nevi. Scale bar = 100 μm.
      Figure thumbnail fx10
      Supplementary Figure S10In vivo cellular characterization of day 30 lCMN xenografts. (a) Experimental procedure: day 1 and day 30 lCMN tissue xenografts injected with or without inhibitors combination (binimetinib and GSK690693) and harvested respectively on day 1 and day 30 postinjections. (b, c) Immunofluorescent stainings and histograms for (b) MelanA and (c) Sox10 in epidermis and dermis. MelanA+ cells were stained using Alexa488-conjugated antibodies. Sox10+ cells were stained using Cy3-conjugated antibodies. (d) Immunohistochemical stainings for phosphoMEK1/2(S221), phosphoERK1/2(T202/Y204), and phosphoGSK-3β(S9) in epidermis and dermis. Phospho-MEK1/2 and phospho-ERK1/2 positive cells were stained using AEC chromogen. n = 3; 2 NRASQ61R + 1 NRAS Q61K. GSK, glycogen synthase kinase; ERK, extracellular signal–regulated kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/ERK kinase. Scale bar = 100 μm.
      Figure thumbnail fx11
      Supplementary Figure S11In vivo cellular characterization of day 1 lCMN xenografts. (a, b, d) Immunofluorescent stainings of day 1 lCMN tissue xenografts with or without inhibitors with (a) K14/Ki67, (b) cleaved-caspase 3, and (d) human mitochondria in epidermis and dermis. (c) Histograms for cleaved caspase-3+ cells in epidermis and dermis. K14+ and cleaved-caspase 3+ cells were stained using Alexa488-conjugated antibodies. Ki67+ and human mitochondria+ cells were stained using Cy3-conjugated antibodies. n = 3; 1 NRASQ61K + 1 NRAS Q61R + 1 NRAS Q61L. lCMN, large congenital melanocytic nevi. Scale bar = 100 μm.
      Figure thumbnail fx12
      Supplementary Figure S12In vivo cellular characterization of day 1 normal skin xenografts. Day 1 normal skin xenografts injected with or without inhibitors (n = 3 from 3 donors). (a) Hematoxylin and eosin (upper panels), phosphoMEK1/2(S221), phosphoERK1/2(T202/Y204), and phosphoGSK-3β(S9) (lower panels) stainings. (b) Measurement of epidermal thickness of day 1 normal skin xenografts injected with or without inhibitors. (c) Immunostainings and histograms for MelanA (left panels), Sox10/Ki67 (middle panels), and K14/Ki67 (right panels). PhosphoMEK1/2+, phosphoERK1/2+, and phosphoGSK-3β+ cells were stained using AEC chromogen. MelanA+, Sox10+, and K14+ cells were stained using Alexa488-conjugated antibodies. Ki67+ cells were stained using Cy3-conjugated antibodies. Error bars: mean ± SEM. ERK, extracellular signal–regulated kinase; GSK, glycogen synthase kinase; lCMN, large congenital melanocytic nevi; MEK, mitogen-activated protein kinase/ERK kinase. Scale bar = 100 μm.
      Figure thumbnail fx13
      Supplementary Figure S13In vivo histological characterization of main organs harvested on day 1 postinjections. Hematoxylin and eosin stainings of heart (upper panels), liver (middle panels), and kidney (lower panels) of mice injected with or without inhibitors. Scale bar = 100 μm.
      Supplementary Table S1All patients included in the study harbor NRAS-mutated CMN
      PatientAgeSexBody SitePAS (cm)Krengel ClassificationBRAF
      Pyrosequencing was used to screen NRAS exon 2 and 3 and BRAF exon 15 mutations.
      Sequencing
      NRAS
      Pyrosequencing was used to screen NRAS exon 2 and 3 and BRAF exon 15 mutations.
      Sequencing
      14 moFHead20-30Large L1WTNRAS3 Q61K
      28 moFHead20-30Large L1WTNRAS3 Q61K
      318 moMHead20-30Large L1WTNRAS3 Q61R
      426 moMArm30-40Large L2WTNRAS3 Q61K
      54 moFTrunk30-40Large L2WTNRAS3 Q61R
      68 yFHead20-30Large L1WTNRAS3 Q61K
      716 moFLeg20-30Large L1WTNRAS3 Q61K
      82.5 yMHead20-30Large L1WTNRAS3 Q61K
      94 yFTrunk30-40Large L2WTNRAS3 Q61K
      104 moFTrunk20-30Large L1WTNRAS3 Q61K
      116.5 yMTrunk40-60Giant G1WTNRAS3 Q61K
      125 yFTrunk30-40Large L2WTNRAS3 Q61K
      134 yFTrunk30-40Large L2WTNRAS3 Q61L
      145 moFTrunk20-30Large L1WTNRAS3 Q61K
      1512 moFHead20-30Large L1WTNRAS3 Q61R
      1611 moMHead20-30Large L1WTNRAS3 Q61K
      174.5 yFTrunk30-40Large L2WTNRAS3 Q61R
      Mutation enrichment by Enhanced-ice-COLD-PCR was also used to screen BRAF exon 15 and NRAS exon 3 mutations.
      Abbreviations: F, female; M, male; mo, month; WT, wild type; y, year.
      1 Pyrosequencing was used to screen NRAS exon 2 and 3 and BRAF exon 15 mutations.

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      Linked Article

      • Can Combination MEK and Akt Inhibition Slay the Giant Congenital Nevus?
        Journal of Investigative DermatologyVol. 139Issue 9
        • Preview
          The clinical management of large and giant congenital melanocytic nevi (lgCMN) relies heavily upon iterative surgical procedures. In this issue Rouille et al. (2019) use lgCMN explants and a newly developed patient-derived xenograft model to show that the local administration of MEK and Akt inhibitors limits the lgCMN proliferative potential. These findings, along with emerging reports, support continued investigation of targeted therapies in lgCMN.
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